Sphingosine-1-Phosphate Receptor Agonist, FTY720, Restores Coronary Flow Reserve in Diabetic Rats

Abstract

Background: Impairment of coronary flow reserve (CFR) has been generally demonstrated in diabetic patients and animals with microvascular complications but without obvious obstructive coronary atherosclerosis. There have been few studies investigating CFR in cases of relatively well-controlled therapy. The purpose of this study is to evaluate the effect of treatment with a Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, on early diabetic rats in terms of CFR.

Methods and Results: Male Sprague-Dawley (SD) rats were divided into 3 groups: (1) streptozotocin-uninjected rats (control rats); (2) streptozotocin-injected hyperglycemic rats (diabetic group); and (3) FTY720-fed and streptozotocin-injected hyperglycemic rats. FTY720 (1.25 mg/kg per day orally) was administrated for 9 weeks in SD rats (from 6 weeks old to 15 weeks old). CFR was evaluated by ¹³NH₃-positron emission tomography. No obvious pathological changes of macrovascular atherosclerosis were observed in each group. Diabetic rats had impaired CFR compared with the control group (1.39±0.26 vs. 1.94±0.24, P<0.05). Treatment with FTY720 for 9 weeks attenuated the heart histological changes and improved CFR in 32% of diabetic rats (1.84±0.36 vs. 1.39±0.26, P<0.05).

Conclusions: In summary, long-term therapy with the Sphingosine-1-phosphate receptor agonist, FTY720, improved CFR by attenuating the heart histological changes, and it might have a beneficial effect on coronary microvascular function in diabetic rats. (Circ J 2014; 78: 2979–2986)

Coronary flow reserve (CFR) is a measure of the coronary vascular functionality and could be an important functional parameter to understand the pathophysiology of coronary microcirculation.¹ Impairment of CFR has been generally demonstrated in diabetic patients and animals with microvascular complications but without obvious obstructive coronary atherosclerosis.^2,3 Impaired CFR has been shown to confer great prognostic information for hard cardiovascular outcome, including all-cause mortality, cardiovascular death and non-fatal myocardial infarction.^4,5

FTY720, as a synthesized Sphingosine-1-phosphate (S1P) receptor agonist, can lower the inflammation reaction and promote lymph cell homing,^6,7 and has an important cardioprotective effect. Recently, it has been found that FTY720 has an important protect effect for diabetic microvessels and macrovessels.^8–11 To date, no study has found an improvement in CFR due to long-term FTY720 administration.

Positron emission tomography (PET) is one of the few non-invasive methods available to quantitatively evaluate coronary vasomotor function.^12–14 By using PET with ¹³N-ammonia, it is possible to measure myocardial perfusion in a non-invasive way and quantify myocardial blood flow (MBF) in a resting and pharmacologic stress phase with adenosine or dipyridamole (hyperemic response).¹⁵ The use of ¹³N-ammonia for quantification of MBF has been compromised by the non-linear relationship between tracer tissue retention and myocardial blood flow, and is suitable for the characterization of the status of coronary arteries and measuring the coronary perfusion reserve non-invasively.

The purpose of the present study was to determine whether impaired coronary microvascular function in diabetic rats could be improved by the long-term administration of FTY720. We used the ¹³NH₃ MicroPET technique for detecting myocardial function in vivo to investigate the impact of CFR in the early stage of diabetes.

Methods

Materials

All reagents, unless otherwise specified, were of analytical grade and commercially available, and were used as received without further purification. FTY720 and streptozotocin were purchased from Sigma-Aldrich Inc (St. Louis, MO, USA). A MicroPET system (Inveon, Siemense Inc, Erlangen, Germany) supplied by Jiangsu Institute of Nuclear Medicine was used for in vivo imaging.

All of the animal experiments were evaluated and approved by the Animal and Ethics Review Committee of Jiangsu Institute of Nuclear Medicine. Experiments were performed in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines on animal research.¹⁶ All efforts were made to minimize the number of animals used and reduce their suffering.

Animals

Male Sprague-Dawley rats (Shanghai Super-B&K Laboratory Animal Corp Ltd, Shanghai, China), weighing 160–180 g (5 weeks old) at the beginning of the study, received a single intraperitoneal injection of streptozotocin (60 mg/kg, dissolved in 0.1 mol/L sodium citrate buffer, pH=4.5) (Sigma) to induce high glucose. Control animals received only the citrate buffer. Only rats that developed sustained hyperglycemia with a serum glucose level of 300 mg/dl were included in the study. After confirmation of diabetes mellitus (DM), rats were divided randomly into 3 groups: (1) streptozotocin-uninjected rats (control group, n=8); (2) streptozotocin-injected hyperglycemic rats (diabetic group, n=8); and (3) FTY720-fed and streptozotocin-injected hyperglycemic rats (FTY720-treated group, n=8). To examine the effect of FTY720 on heart microvessels, we fed diabetic rats with or without oral FTY720 administration (with an effective dose of 1.25 mg·kg^–1·day^–1) for a duration of 9 weeks.

The animals were maintained in a room controlled at a temperature range of 20–26˚C and relative humidity of 40–70%. The weight and serum glucose level of all groups were tested every 2 weeks during the experiment.

Positron Emission Computed Tomography and Data Analysis

After treated with FTY720 for 9 weeks, all animals underwent the Positron Emission Tomography study at rest and under dipyridamole vasodilatation conditions. First, the rats received an injection of ¹³N-ammonia (3.5–5.0 mCi) in the tail vein. Dynamic serial transaxial images were acquired for 10 min (Figure 1A). The proportion of ¹³N radioactivity in plasma was measured in rats with a catheter inserted in a femoral artery. Blood samples were collected (150 μl) at 30, 60, 120, 180 and 300 s from the femoral artery catheter. After deproteinization and centrifugation, the supernatant was transferred to new tubes. Triplicates of the counting tubes containing the supernatant were counted individually using a gamma counter for 1 min. Sixty minutes after the first ¹³N-ammonia injection, 150 μg·kg^–1·min^–1 dipyridamole (Beijing Yongkang Co) was infused intravenously over 4 min. ¹³N-ammonia (3.5–5.0 mCi) was injected at the end of the third minute after dipyridamole infusion and serial images of the heart were recorded 3 min (Figure 1B). The whole PET study took an average time of 20 min.

Figure 1.

Coronal heart positron emission tomography (PET) perfusion imaging results following intravenous administration of ¹³N-ammonia. (A) ¹³N-ammonia PET heart imaging of a rat at rest. (B) ¹³N-ammonia PET heart imaging of a rat after an injection of dipyridamole.

For imaging studies, animals were under isoflurane anesthesia that was delivered through a nose cone at a concentration of 2.0% volume and 2 L/min oxygen flow. Animals were positioned and immobilized supine, with their medial axis parallel to the axial axis of the scanner, with their thorax region in the center of field of view. Imaging experiments were performed on the Siemens Inveon Dedicated Small Animal PET Scanner, with an in-plane resolution of 0.48 mm and an energy width of 350–650 kev. The images were reconstructed with the size of 128×128 and with an OSEM3D format. The slice thickness was 0.78 mm. The quantitative analysis of the cardiac distribution of ¹³N-ammonia tracer was performed using ASIProVM (version 6.7.1.1).

In our experiment, CFR was defined as the ratio of stimulated myocardial blood flow (stress) to baseline (resting), and it was used to examine the integrity of coronary microvascular circulation. The regions of interest on each myocardial wall were manually drawn on the ¹³N-ammonia PET images taken at baseline and during dipyridamole provocation in each group. A 3-compartment kinetic model was used for the computation of MBF as described in the literature.¹⁷ The model parameter was computed using the blood and tissue curves derived from PET data.

Histopathological Evaluation and Immunohistochemistry

After imaging experiments, the rats were sacrificed and their left ventricles were dissected and myocardial sections were removed immediately for histological processing. The tissue samples were fixed in paraformaldehyde solution (4%) and embedded in paraffin. From the paraffin blocks, 4 μm-thick serial sections were examined for detailed morphological analysis. The heart tissue was estimated by using hematoxylin and eosin (H&E) staining.

All sections of formalin-fixed, paraffin-embedded heart samples for immunohistochemical analysis were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 min at room temperature. The sections were microwaved to retrieve the antigen by using an antigen unmasking solution for 5 min. After incubation with 5% skim milk for 1 h at room temperature, the sections were incubated with the primary antibodies (rabbit anti-rat CD31 (1:100), anti-transforming growth factor (TGF)-β (1:100), anti-collagen type I (1:100) overnight at 4ºC). Then, the tissues were washed several times in phosphate-buffered saline (PBS) and incubated with the secondary antibody for 45 min. Subsequently, the tissues were visualized with a 3, 3’-diaminobenzidine kit and examined under a microscope and photographed.

The collagen distribution in myocardial tissue was observed by Masson’s trichrome staining and collagen immunohistochemistry staining. In order to observe the changes of the capillaries in the myocardium, endothelial cells were stained with anti-CD31. To determine capillary density, the number of CD31 brown positive-staining cells was counted in a double-blind fashion from 10 different fields of each section (n=5) at a ×200 magnification. The average number of the vessels in one section was used for the assessment of microvascular density. All images were reviewed and analysed using Image Pro Plus 6.0.

Western Blotting

We used Western blot analysis according to the manufacturer’s instructions to investigate vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 protein expression. Proteins from the heart tissue extracts (100 μg per sample) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Non-specific antibody binding was blocked by a pre-incubation of the membrane in 1×Tris-buffered saline containing 5% skim milk for 1 h at room temperature. The membranes were incubated with anti-VCAM-1 antibody (1:500; Sigma), anti-IL-6 antibody (1:500; Sigma) and anti-ICAM-1 antibody (1:500; Sigma) at 4ºC overnight with constant shaking. This was followed by incubation with the appropriate peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, the antibody-bound proteins were detected using an ECL advanced system (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK); β-action (1:1,000; Santa Cruz Technology, TX, USA) was used as the internal control protein to normalize the samples.

Statistical Analysis

Data are expressed as mean±standard deviation (SD) and analyzed by using SPSS 17.0 software. For comparisons between 2 variables, the unpaired Student’s t-test was used. A two-tailed P<0.05 was considered statistically significant.

Results

Characteristics of Diabetic Animals

Three rats died during the experimental period; 2 in the vehicle-treated diabetic group and 1 in the FTY720-treated group. Serum levels of glucose were increased in diabetic rats, which were higher than 18 mmol/L in 6 weeks old. These results indicate that hyperglycemia was successfully induced. Interestingly, the blood glucose level in the FTY720-treated group became lower than that in the diabetic group after being treated for 4 weeks, and this lasted until the end of our experiment.

Diabetic rats showed lower body weight, significantly increased urine glucose levels and daily water intake. These parameters were not significantly altered by FTY720 treatment (Table 1). The changes in weight and blood glucose level for each group are shown in Table 1.

Table 1. General Characteristics for the Control, the Streptozotocin-Induced Diabetic Group and the FTY720 Treatment Group

Parameters	Week	Control (n=8)	DM (n=6)	DM+FTY720 (n=7)
Weight (g)	6	170±13.8	168±12.6	172±10.5
	9	355±32.5	242±12.5*	256±13.7
	12	487±98.2	277±14.6*	298±15.8
	15	525±102.9	298±21.8*	309±16.2
Urine glucose (qualitative)	6,9,12,15	Negative	Positive	Positive
Blood glucose (mmol/L)	6	6.8±0.6	19.8±1.4*	20.63±1.6
	9	7.1±0.4	22.12±2.2*	17.23±2.3#
	12	7.3±0.6	25.3±3.10*	17.23±3.5#

Values are presented as mean±SD, *P<0.01 vs. Control, ^#P<0.05 vs. DM. DM, diabetes mellitus; FTY720, Sphingosine-1-phosphate (S1P) receptor potent agonist.

Chronic FTY720 Treatment Restores CFR in Diabetic Animals

Eighteen rats were studied following ¹³N-ammonia injection at baseline and under pharmacologic intervention conditions (Table 2). Although there was no obvious different average MBF value between the 3 groups, under dipyridamole stimulation conditions, diabetic rats had significantly decreased CFR compared with the non-diabetic control group (1.39±0.26 vs. 1.94±0.24, P<0.05). After given FTY720 for 9 weeks, we observed an increase of CFR by 31% compared in the diabetic group (1.84±0.36 vs. 1.39±0.26, P<0.05), indicating that FTY720 preserved the coronary artery diastolic ability.

Table 2. CFR and Perfusion Data at Baseline and During Hyperemia in the Controls, Diabetic and FTY720 Treatment Groups

Parameters	Control (n=6)	DM (n=6)	DM+FTY720 (n=6)
Blood glucose (mmol/L)	10.2±1.8	27.5±2.7*	15.3±2.49#
MBFRest (ml·min−1·g−1)	6.13±1.23	5.96±1.03	6.04±1.37
MBFStress (ml·min−1·g−1)	11.62±1.58	8.35±1.58*	11.14±2.12#
CFR	1.94±0.24	1.39±0.26*	1.84±0.36#

Values are presented as mean±SD. *P<0.05 vs. Control. ^#P<0.05 vs. DM. CFR, coronary flow reserve; MBF, myocardial blood flow. Other abbreviations as in Table 1.

Effect of FTY720 on Histological Changes

Conventional histology with H&E and Masson staining showed moderate myodegeneration and sporadic vacuolization in the left ventricular myocardium of the diabetic control rats, without signs of severe tissue damage, and no obvious pathological changes of large vessel atherosclerosis.

After H&E staining, neat myocardial cells that had a clear structure, were compact, with less extracellular matrix, and a small amount of fibroblasts, could be seen in the normal rats. While in the diabetic group, disorderly arranged myocardial cells, distortion, and enlarged cell gaps were shown. Cardiomyocyte size significantly increased in the diabetic group compared to the control group, while in the FTY720 treatment group, a trend towards diminished cardiomyocyte hypertrophy, but no statistically significant effects, were found in comparison with the control group. The state was somewhat in between, and compared with the model group, narrowed cell gaps and reduced interstitial and perivascular extracellular matrix were shown (Figure 2A).

Figure 2.

A Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, attenuates heart histological changes in experimental diabetic rats. Representative images of hematoxylin and eosin (H&E) (A) and Masson stains (B) showing cardiomyocyte hypertrophy and cardiointerstitial fibrosis. Increased ventricular fibrosis was present in the heart of diabetic rats compared to the control group. These changes were suppressed by treatment with FTY720 (C and D, n=6). ^∆P<0.05 vs. control. *P<0.05 vs. diabetic rats. Magnification ×200 (A and B).

After Masson staining, the distribution of collagen tissue was almost equal in the non-diabetic control group; the collagen fiber network among adjacent cells was intact with less collagen fiber content. In the diabetic group, the broken and disorderly arranged collagen fiber network around myocardial cells increased interstitial and perivascular extracellular matrix, and increased the number of fibroblasts with inflammatory cells infiltrated, as shown in Figure 2B. Compared with the control group, the relative myocardial collagen content in the diabetic rat increased significantly (P<0.01), while in the FTY720 treatment group, the arrangement of collagen tissue and the structure of the fiber network were better (Figure 2B). Compared with the diabetic group, collagen content in the FTY720 treatment group decreased significantly (P<0.05), suggesting that FTY720 can remarkably inhibit the proliferation of collagen fibers.

Effects of FTY720 on the Expression of Collagen and the Pro-Fibrotic Molecule TGF-β

Transforming growth factor (TGF)-β plays an important role in the pathogenesis of the fibrotic effects in diabetic complications. Immunohistochemical staining for TGF-β demonstrated a similar pattern to that observed with respect to collagen content. As shown in Figure 3C, diabetic rats had significantly increased TGF-β immunostaining compared with the control rats (5.66±0.92% vs. 3.22±0.56%, P<0.05). TGF-β immunostaining in the FTY720-treated diabetic group was significantly lower than that in the vehicle-treated diabetic rats (4.42±0.29% vs. 5.66±0.92%, P<0.05), but significantly higher compared with the control rats (P<0.05). To assess the extent of myocardial interstitial fibrosis, we measured the collagen content by collagen staining the sections of the left ventricle from experimental animals. As shown in Figure 3B, quantitative analysis of the fibrotic region of the left ventricle myocardium indicated significantly increased interstitial fibrosis in DM animals vs. control (11.7±0.87% vs. 6.21±0.67%, P<0.05). FTY720 treatment significantly reduced the extent of myocardial interstitial fibrosis compared with the diabetic rats (7.96±0.80% vs. 11.7±0.87%, P<0.05), but it was significantly higher compared with the control rats (P<0.01).

Figure 3.

Effect of a Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, on transforming growth factor (TGF)-β and collagen expression in the heart tissue of diabetic rats. Typical results of TGF-β and collagen protein expression by immunohistochemical 3, 3’-diaminobenzidine (DAB) staining. Values are represented as mean±SD (n=6). ^∆P<0.05 vs. control. *P<0.05 vs. diabetic rats.

Effects of FTY720 on Ventricle Capillary Density

The myocardial microvessel density difference was quantitative assessed by using CD31-positive staining endothelial cells. Endothelial cells in coronary microvasculature were regularly arranged, and the capillary walls were thin and smooth in most control rats (Figure 4A). However, there was a significant decrease of cardiac capillary density in the diabetic group compared to the control group (11.4±0.79% vs. 22.54±3.59%, P<0.01). FTY720 treatment for 9 weeks significantly ameliorated the damage of the microvasculature and a trend towards increased capillary density was found compared to the control group (17.26±1.40% vs. 11.4±0.79%, P<0.05).

Figure 4.

A Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, increases the number of CD31+ endothelial cells in the tissue of diabetic rats. (A) CD31 expression in the heart ventricular capillary endothelium (original magnification ×200, 3, 3’-diaminobenzidine (DAB) staining). (B) Density of the ventricular capillary (per 0.065 mm², n=6). ^∆P<0.05 vs. control. *P<0.05 vs. diabetic rats.

Effects of FTY720 on the Expression of Pro-Inflammatory and Endothelial Adhesion Molecule Expression

As shown in Figure 5, Western blot analysis results show that the diabetic group had a significant increase of IL-6 in cardiac tissues, while in the FTY720 treatment group, the level of IL-6 was lower than that of the DM group. Compared to controls, diabetic rats had significantly increased VCAM-1 and ICAM-1 protein relative levels (VCAM-1 protein: 0.29±0.04 vs. 0.12±0.03; ICAM-1 protein: 0.78±1.02 vs. 0.39±0.05, P<0.01). However, FTY720 treatment of diabetic rats resulted in significantly decreased VCAM-1 and ICAM-1 protein levels compared with the untreated diabetic rats (VCAM-1 protein: 0.18±0.04 vs. 0.29±0.04; ICAM-1 protein: 0.59±0.07 vs. 0.78±1.02, P<0.05).

Figure 5.

Effect of a Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, on interleukin (IL)-6 and on the Endothelial Adhesion Molecule Expression in the heart tissue of diabetic rats. (A) Western blotting analysis for IL-6, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1. (B) Relative protein expression of IL-6, VCAM-1 and ICAM-1. Values are represented as the mean±SD (n=3). *P<0.05 vs. control. ^#P<0.05 vs. diabetic rats.

Discussion

In the present study, we successfully constructed the diabetic rat model and studied the effect of FTY720 on CFR with ¹³N-ammonia microPET. Our experimental results showed that there was a lower flow coronary vasodilator response in the majority of diabetic rats. After oral administration of FTY720, at a dose of 1.25 mg·kg^–1·day^–1 for 9 weeks, the CFR of diabetic rats increased. It indicated that FTY720 improves the vasodilator response of myocardial microvessels. To the best of our knowledge, this is the first report on the administration of FTY720, which prevented the progression of coronary microcirculatory disturbance.

Evidence from clinical and experimental studies showed that CFR is reduced in diabetic mellitus because of functional and structural abnormalities of the coronary microvascular circulation such as endothelial dysfunction, hyperglycemia, perivascular fibrosis, endothelial adherence molecules and collagen disposition.¹⁸ An abnormal extravascular component of coronary resistance, like smooth muscle cell proliferation, might also play an important role.¹⁹ A previous study has shown that inward hypertrophic remodeling of coronary arterioles isolated from diabetic mice is associated with a decrease in vascular wall stiffness and reduced MBF and CFR.²⁰ In this study, our findings were consistent with the current literature and showed that FTY720 treatment lowered the expression of TGF-β and collagen, and reduced the extent of myocardial interstitial fibrosis in diabetic rat heart tissues.

Sphingosine-1-phosphate receptor agonists have a wide effect on the proliferation of microvessels and inhibited the apoptosis of vessel endothelial cells.²¹ Some studies have shown that S1P could control the vascular tone;^22,23 however, there are few studies that examined the effect of the S1P receptor agonist on the impact of myocardial perfusion. As one type of selective S1P receptor agonist drug, FTY720 has been proven to be of low toxicity. This study revealed that after 3 weeks of FTY720 treatment, there was no impact on kidney and liver hemodynamics.²⁴ Our ¹³N-NH₃ PET animal myocardial perfusion experiment indicated that the oral administration of FTY720 for 9 weeks could restore the CFR of diabetes rats. And pathology experiment showed that long-term administration of FTY720 improves myocardial microangiopathy.

There has been controversy about the effect of S1P agonist on the expression of vascular inflammation factor and endothelial adhesion molecule. Some studies support the use of the S1P receptor agonist to prevent inflammation factor-mediated monocyte adhesion to aortic endothelium in mice,^25,26 while another study showed that S1P could induce an increase in E-selectin expression by the way of NF-κB activation.²⁷ However, the present study results showed that FTY720 could lower the expression of VCAM-1, ICAM-1 and IL-6 in diabetic rat heart tissue. A recent study report that IL-6 and tumor necrosis factor (TNF)-α were independently associated with CFR,²⁸ and a PET-CFR study showed that IL-6 should be a determinant of CFR.²⁹ This may be one of the microvascular protection mechanisms of FTY720. Interestingly, the FTY720 treatment group showed a lower glucose level than the diabetic group; one of the mechanisms of FTY720 treatment may be that it could prevent the manifestation of DM by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and β-cell destruction by proinflammatory cytokines.^30–32 This protective effect might account for why FTY720 could restore the CFR of diabetic rats and improve vascular resistance.

Diabetes could be a coronary artery disease equivalent.^33,34 The earlier stage of diabetes impacts the vascular system and causes endothelial dysfunction, such as the increase of reactive oxygen species, the lower release of NO, and the stimulation of endothelial cells to release a vascular inflammation factor and bring about the state of apoptosis. This procedure is also called “endothelial activation”, which reflects an early injury procedure to the vascular wall, and could lead to the disturbance of the microvascular blood stream and the lower myocardial perfusion.³⁵ The coronary microvascular endothelial dysfunction has an important role in the development of atherosclerosis. Some studies have demonstrated that FTY720 could promote angiogenesis, lower the vascular permeability,³⁶ stimulate endothelial cells to release NO by the PI3K/AKT pathway and increase the local blood flow.^37,38 Another study has shown that endothelium-derived hyperpolarizing factor (EDHF) dysregulation is present in various vascular beds of diabetic animal models and humans with vascular dysfunction caused by an impairment of endothelial-dependent vasodilation; EDHF also plays a crucial role in modulating vasomotor tone in microvessels.³⁹ How FTY720 influences the endothelium-derived relaxing factor and the mechanism that impacts the endothelial function remains unclear.

Overall, there is growing evidence that lower CFR is an independent risk factor of cardiovascular events.^40,41 Therefore, study drugs to improve coronary artery microcirculation have an important clinical value. Myocardial ¹³N-NH₃ PET imaging provides a relatively precise method for evaluating coronary microcirculation. We used this method to study the effect of FTY720 on the restored CFR of the diabetic rat. This reverse effect might have important implications for coronary microcirculation, but the mechanism is complex and future research is needed. In this study, the animal model was given a large dose of streptozotocin and was not provided with high cholesterol food to feed on; this was the type 1 diabetic model. CFR is affected in the experimental rats not only by microvascular function but also by epicardial coronary stenosis.⁴² However, in this study, we did not find obvious pathogenesis of macrovascular complications, including atherosclerosis in diabetic rats. Whether FTY720 could restore the CFR, which deteriorated because of coronary macrovessel stenosis, is still unknown. The next step in our research would be to use a low dose injection of streptozotocin (30 mg/kg) along with a high-fat diet to induce high blood pressure and high cholesterol levels to study the effect of FTY720 on CFR in a diabetic model that has obstructive coronary atherosclerosis.

Study Limitations

The main drawback of this study is the high cost and the limited availability of the imaging equipment in some places throughout the world. However, PET has recently proved itself as a highly precise method for the assessment of CFR. In addition, these results will reinforce PET’s key role in cardiology and help to make it a more accessible method in the future. Other limitations of this study include a low number of rats used and the short length of FTY720 treatment. One of the most important limitations of this study is the fact that we were not able to compare the different dose effects of the drugs separately, thus we cannot conclude whether the changes observed were due mostly to the effectiveness of the drugs. It is important to design a study that includes a larger number of doses given to the subjects, a longer treatment course, and to create groups that could receive each of the drugs independently. In addition, some studies report that FTY720 has side-effects such as transient bradycardia and atrioventricular block. Some studies reported that heart rate and blood pressure can impact CFR.⁴³ Further research is required to investigate whether FTY720 has an impact on the heart rate of diabetic rats.

Acknowledgments

This work was supported by the Open Program of Key Laboratory of Nuclear Medicine, Ministry of Health and Jiangsu Key Laboratory of Molecular Nuclear Medicine (No. KF201302). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the staff of Wuxi Molecular PET Centre for their excellent technical assistance.

Disclosures

The authors have declared that no competing interests exist.

Appendix

The contributions of the authors to this study were as follows:

Conceived and designed the experiments: H.X., H.N., Y.J.

Performed the experiments: H.X., H.N., S.H.

Analyzed the data: H.N., S.H.

Wrote the paper: H.X., H.N.

Sponsored the experiments: Y.J.

References

• 1.    Matsuo  H. Microvascular dysfunction: Clinically relevant but still difficult to detect. Circ J 2013; 77: 1687–1688.
• 2.    Atar  AI,  Altuner  TK,  Bozbas  H,  Korkmaz  ME. Coronary flow reserve in patients with diabetes mellitus and prediabetes. Echocardiography 2012; 29: 634–640.
• 3.    Nemes  A,  Forster  T,  Geleijnse  ML,  Kutyifa  V,  Neu  K,  Soliman  OI, et al. The additional prognostic power of diabetes mellitus on coronary flow reserve in patients with suspected coronary artery disease. Diabetes Res Clin Pract 2007; 78: 126–131.
• 4.    Tsukamoto  O,  Kitakaze  M. Reserve of coronary flow deserves predictor of cardiovascular events. Circ J 2012; 76: 1834–1835.
• 5.    Sheth  T,  Yusuf  S. Enhancing risk prediction with PET coronary flow reserve: Can it be clinically useful? JACC Cardiovasc Imaging 2012; 5: 1035–1036.
• 6.    Matloubian  M,  Lo  CG,  Cinamon  G,  Lesneski  MJ,  Xu  Y,  Brinkmann  V, et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004; 427: 355–360.
• 7.    Mandala  S,  Hajdu  R,  Bergstrom  J,  Quackenbush  E,  Xie  J,  Milligan  J, et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 2002; 296: 346–349.
• 8.    Yin  Z,  Fan  L,  Wei  L,  Gao  H,  Zhang  R,  Tao  L, et al. FTY720 protects cardiac microvessels of diabetes: A critical role of S1P1/3 in diabetic heart disease. PLoS One 2012; 7: e42900, doi:10.1371/journal.pone.0042900.
• 9.    Whetzel  AM,  Bolick  DT,  Srinivasan  S,  Macdonald  TL,  Morris  MA,  Ley  K, et al. Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor. Circ Res 2006; 99: 731–739.
• 10.    Nofer  JR,  Bot  M,  Brodde  M,  Taylor  PJ,  Salm  P,  Brinkmann  V, et al. FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development of atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 2007; 115: 501–508.
• 11.    Keul  P,  Tolle  M,  Lucke  S,  von Wnuck Lipinski  K,  Heusch  G,  Schuchardt  M, et al. The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2007; 27: 607–613.
• 12.    Tamaki  N,  Yoshinaga  K,  Naya  M. Coronary vasomotor function assessed by positron emission tomography. Eur J Nucl Med Mol Imaging 2010; 37: 1213–1224.
• 13.    Schindler  TH,  Zhang  XL,  Vincenti  G,  Mhiri  L,  Lerch  R,  Schelbert  HR. Role of PET in the evaluation and understanding of coronary physiology. J Nucl Cardiol 2007; 14: 589–603.
• 14.    Schindler  TH,  Schelbert  HR,  Quercioli  A,  Dilsizian  V. Cardiac PET imaging for the detection and monitoring of coronary artery disease and microvascular health. JACC Cardiovasc Imaging 2010; 3: 623–640.
• 15.    Nitzsche  EU,  Choi  Y,  Czernin  J,  Hoh  CK,  Huang  SC,  Schelbert  HR. Noninvasive quantification of myocardial blood flow in humans: A direct comparison of the [13N]ammonia and the [15O]water techniques. Circulation 1996; 93: 2000–2006.
• 16.    Kilkenny  C,  Browne  WJ,  Cuthill  IC,  Emerson  M,  Altman  DG. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol 2010; 8: e1000412, doi:10.1371/journal.pbio.1000412.
• 17.    Croteau  E,  Benard  F,  Bentourkia  M,  Rousseau  J,  Paquette  M,  Lecomte  R. Quantitative myocardial perfusion and coronary reserve in rats with 13N-ammonia and small animal PET: Impact of anesthesia and pharmacologic stress agents. J Nucl Med 2004; 45: 1924–1930.
• 18.    Ahmed  B. New insights into the pathophysiology, classification, and diagnosis of coronary microvascular dysfunction. Coron Artery Dis 2014; 5: 439–449.
• 19.    Camici  PG,  Olivotto  I,  Rimoldi  OE. The coronary circulation and blood flow in left ventricular hypertrophy. J Mol Cell Cardiol 2012; 52: 857–864.
• 20.    Katz  PS,  Trask  AJ,  Souza-Smith  FM,  Hutchinson  KR,  Galantowicz  ML,  Lord  KC, et al. Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct pattern of remodeling associated with decreased vessel stiffness. Basic Res Cardiol 2011; 106: 1123–1134.
• 21.    Lee  MJ,  Thangada  S,  Claffey  KP,  Ancellin  N,  Liu  CH,  Kluk  M, et al. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 1999; 99: 301–312.
• 22.    Dantas  AP,  Igarashi  J,  Michel  T. Sphingosine 1-phosphate and control of vascular tone. Am J Physiol Heart Circ Physiol 2003; 284: H2045–H2052.
• 23.    Peters  SL,  Alewijnse  AE. Sphingosine-1-phosphate signaling in the cardiovascular system. Curr Opin Pharmacol 2007; 7: 186–192.
• 24.    Brinkmann  V,  Cyster  JG,  Hla  T. FTY720: Sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am J Transplant 2004; 4: 1019–1025.
• 25.    Bolick  DT,  Srinivasan  S,  Kim  KW,  Hatley  ME,  Clemens  JJ,  Whetzel  A, et al. Sphingosine-1-phosphate prevents tumor necrosis factor-{alpha}-mediated monocyte adhesion to aortic endothelium in mice. Arterioscler Thromb Vasc Biol 2005; 25: 976–981.
• 26.    Wang  F,  Okamoto  Y,  Inoki  I,  Yoshioka  K,  Du  W,  Qi  X, et al. Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice. J Clin Invest 2010; 120: 3979–3995.
• 27.    Wang  L,  Xing  XP,  Holmes  A,  Wadham  C,  Gamble  JR,  Vadas  MA, et al. Activation of the sphingosine kinase-signaling pathway by high glucose mediates the proinflammatory phenotype of endothelial cells. Circ Res 2005; 97: 891–899.
• 28.    Tona  F,  Serra  R,  Di Ascenzo  L,  Osto  E,  Scarda  A,  Fabris  R, et al. Systemic inflammation is related to coronary microvascular dysfunction in obese patients without obstructive coronary disease. Nutr Metab Cardiovasc Dis 2014; 24: 447–453.
• 29.    Vaccarino  V,  Khan  D,  Votaw  J,  Faber  T,  Veledar  E,  Jones  DP, et al. Inflammation is related to coronary flow reserve detected by positron emission tomography in asymptomatic male twins. J Am Coll Cardiol 2011; 57: 1271–1279.
• 30.    Moon  H,  Chon  J,  Joo  J,  Kim  D,  In  J,  Lee  H, et al. FTY720 preserved islet beta-cell mass by inhibiting apoptosis and increasing survival of beta-cells in db/db mice. Diabetes Metab Res Rev 2013; 29: 19–24.
• 31.    Jorns  A,  Rath  KJ,  Terbish  T,  Arndt  T,  Meyer Zu Vilsendorf  A,  Wedekind  D, et al. Diabetes prevention by immunomodulatory FTY720 treatment in the LEW.1AR1-iddm rat despite immune cell activation. Endocrinology 2010; 151: 3555–3565.
• 32.    Tsuji  T,  Yoshida  Y,  Fujita  T,  Kohno  T. Oral therapy for type 1 diabetes mellitus using a novel immunomodulator, FTY720 (fingolimod), in combination with sitagliptin, a dipeptidyl peptidase-4 inhibitor, examined in non-obese diabetic mice. J Diabetes Investig 2012; 3: 441–448.
• 33.    Hermans  MP,  Brotons  C,  Elisaf  M,  Michel  G,  Muls  E,  Nobels  F. Optimal type 2 diabetes mellitus management: The randomised controlled OPTIMISE benchmarking study: Baseline results from six European countries. Eur J Prev Cardiol 2013; 20: 1095–1105.
• 34.    Bulugahapitiya  U,  Siyambalapitiya  S,  Sithole  J,  Idris  I. Is diabetes a coronary risk equivalent? Systematic review and meta-analysis. Diabet Med 2009; 26: 142–148.
• 35.    Utriainen  T,  Nuutila  P,  Takala  T,  Vicini  P,  Ruotsalainen  U,  Ronnemaa  T, et al. Intact insulin stimulation of skeletal muscle blood flow, its heterogeneity and redistribution, but not of glucose uptake in non-insulin-dependent diabetes mellitus. J Clin Invest 1997; 100: 777–785.
• 36.    Sanchez  T,  Estrada-Hernandez  T,  Paik  JH,  Wu  MT,  Venkataraman  K,  Brinkmann  V, et al. Phosphorylation and action of the immunomodulator FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability. J Biol Chem 2003; 278: 47281–47290.
• 37.    Rikitake  Y,  Hirata  K,  Kawashima  S,  Ozaki  M,  Takahashi  T,  Ogawa  W, et al. Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenesis. Arterioscler Thromb Vasc Biol 2002; 22: 108–114.
• 38.    Takuwa  Y,  Du  W,  Qi  X,  Okamoto  Y,  Takuwa  N,  Yoshioka  K. Roles of sphingosine-1-phosphate signaling in angiogenesis. World J Biol Chem 2010; 1: 298–306.
• 39.    Gao  X,  Martinez-Lemus  LA,  Zhang  C. Endothelium-derived hyperpolarizing factor and diabetes. World J Cardiol 2011; 3: 25–31.
• 40.    Nakanishi  K,  Fukuda  S,  Shimada  K,  Miyazaki  C,  Otsuka  K,  Maeda  K, et al. Impaired coronary flow reserve as a marker of microvascular dysfunction to predict long-term cardiovascular outcomes, acute coronary syndrome and the development of heart failure. Circ J 2012; 76: 1958–1964.
• 41.    Uchida  Y,  Ichimiya  S,  Ishii  H,  Kanashiro  M,  Watanabe  J,  Yoshikawa  D, et al. Impact of metabolic syndrome on various aspects of microcirculation and major adverse cardiac events in patients with ST-segment elevation myocardial infarction. Circ J 2012; 76: 1972–1979.
• 42.    Gan  LM,  Wikstrom  J,  Fritsche-Danielson  R. Coronary flow reserve from mouse to man: From mechanistic understanding to future interventions. J Cardiovasc Transl Res 2013; 6: 715–728.
• 43.    Forte  EH,  Rousse  MG,  Lowenstein  JA. Target heart rate to determine the normal value of coronary flow reserve during dobutamine stress echocardiography. Cardiovasc Ultrasound 2011; 9: 10.